Work in our laboratory spanning more than two decades has demonstrated that certain drugs may be attached to well-defined carrier molecules and still retain the ability to bind to the receptor site and induce biological activity. This synthetic strategy for the attachment of drugs to carriers is termed the functionalized congener approach. The carrier molecule may be many times larger than the parent drug; indeed there is practically no maximum size limitation for a fully potent analog. Unlike the prodrug approach or the immobilization of drugs for slow release, the functionalized congener approach is designed to produce analogs for which no metabolic cleavage step is necessary for activation. Moreover, the attachment of the drug to a carrier such as a peptide may result in enhanced affinity at an extracellular receptor site and an improvement in the pharmacological profile of the parent drug through energetically favorable interaction with distal sites on a receptor, as modeled for our high affinity fluorescent A2A and P2Y14 receptor antagonists. The recently determined X-ray structures of the adenosine receptors (ARs) and P2Y receptors (P2YRs) for nucleotides follow upon a two-decade long progression of knowledge of the AR binding site(s) and other structural features, based on empirical probing of structure activity relationships (SARs) of ligands, molecular modeling, and mutagenesis analysis. Early modifications of both AR agonists and alkylxanthine antagonists located sites in these two ligand scaffolds that were amenable to extension through a chemically functionalized chain without losing the ability to bind to the ARs. This logically suggested that the putative binding sites had regions that were accessible to the external environment and therefore less demanding sterically, i.e. the functionalized chains protruded beyond the conformationally and sterically restricted binding region of the core pharmacophore. Points on the adenosine scaffold that displayed flexibility of substitution and could be extended chemically, without a limiting length, included the N6 position for the A1AR and the C2 position for the A2AAR.Both of these positions on adenosine derivatives are predicted by receptor docking to point toward the extrallular loops of the receptor. The C2-extended derivatives are most useful in the design of fluorescent agonists of the A2A receptor. In the xanthine series, such modification of the C8 position was relatively insensitive in receptor binding, and thus served as a suitable site for derivatization in AR antagonist functionalized congeners. At the P2Y6R, which is a potential target for diabetes treatment, the nucleobase C4 position is suitable for chain extension (through an alkoxyimino linkage). At the P2Y14R, which is a potential target for treatment of inflammation, the glucose moiety of native agonist UDP-glucose is suitable for chain extension (through an amide linkage to glucuronate). We used both of these points of substitution to design high affinity fluorescent ligands, which have advantages over radioligands as tools in drug discovery. We have used these fluorescent ligands for compound screening by flow cytometry. The fluorescent P2Y14 antagonist MRS4174 is highly potent and has become our standard screening tool in our drug discovery efforts on that receptor. A terminal amino group of the high affinity AR antagonist XAC (a xanthine amine congener) served as a site for generalized coupling to much larger moieties that extended into the extracellular medium, without losing AR affinity. The recently determined X-ray structures of adenosine and XAC (Dore et al., 2011) bound to a thermostabilized hA2AAR show that the terminal amino chain of XAC proved to be very flexible and can be anchored to the receptor in various conformations. Thus, the objective in the design of XAC as a functionalized congener, in which the distal amino group escapes the steric constraints of the pharmacophore binding site, was finally explained structurally. XAC congeners have been incorporated into peptide nucleic acids (PNAs) as spatially defined multivalent probes for exploring the phenomenon of receptor dimerization and oligomerization. Such aggregates of receptors can have their own biological effects, distinct from the monomeric receptors. Ligands for dimeric receptors are largely lacking in the current chemical toolbox. We have explored other multivalent carriers to study GPCR action. For example, dendrimers are tree-like polymers that have multiple functional sites on the periphery for attachment of ligands. We have extensively explored tree-like polyamidoamine (PAMAM) dendrimers as nanocarriers for functionalized congeners, i.e. strategically derivatized ligands for tethering, for ARs and P2YRs. We have already shown that such multivalent conjugates of AR agonists, optionally bearing PEG groups for increasing water solubility, bound to the receptor and displayed antithrombotic activity (A2AAR) or cardioprotective activity (A3AR). However, we note with caution that terminal-amino PAMAM dendrimers undergo chemical rearrangements that make them less than optimal as drug carriers. Gold nanoparticles (AuNPs) allow the tuning of pharmacokinetic and pharmacodynamic properties by active or passive targeting of drugs for cancer and other diseases. We have functionalized AuNPs by tethering specific ligands, agonists and antagonists, of ARs to the gold surface as models for cell surface interactions with their receptors. The AuNP conjugates with chain-extended AR ligands alone (PEGylated nucleosides and nonnucleosides, anchored to the Au suface via thioctic acid) were found to be insoluble in water due to hydrophobic entities in the ligand. Therefore, we added a second, biologically inactive pendant moiety to increase the water solubility, consisting of a PEGylated chain terminating in a carboxylic or phosphate group and found that receptor affinity of the intact conjugates could be retained. Thus, we have synthesized stable, water-soluble AuNP derivatives of tethered A3 and A2AAR ligands intended for therapeutic and imaging applications, which display in vitro biological properties similar to their monomeric ligands. This is the first prototypical application to gold carriers of small molecule (nonpeptide) GPCR ligands, which are under investigation for treatment of cancer and inflammatory diseases. We have also made biologically active conjugates of functionalized congeners of AR ligands attached covalently to quantum dots. It is becoming apparent that the receptors have higher degrees of organization, i.e. they form functionally complex aggregates, and there is a need for pharmacological tools that address this multiplicity. We synthesized fluorescent A2AR agonists and antagonists and evaluated, by means of a flow cytometry homogeneous no-wash assay and a real-time fluorescence resonance energy transfer (FRET)-based approach, the effects on A2AR binding of distinct antiparkinsonian drugs in current clinical use (i.e. pramipexole, rotigotine and apomorphine). Our results provided evidence for the existence of a differential dopamine D2R-mediated negative allosteric modulation on A2AAR agonist binding that was oligomer-formation dependent, and with apomorphine being the best antiparkinsonian drug attenuating A2AAR agonist binding. Overall, the here-developed methods were found valid to explore the ability of drugs acting on D2Rs to modulate A2AAR binding, thus serving to facilitate the preliminary selection of D2R-like candidate drugs in the management of Parkinsons disease. Striking differences have been noted between the monomeric ligands and the multivalent dendrimer conjugates - either in the potency or selectivity of the ligand or in the kinetics of the biological effect.